1. Field of the Invention
The invention is drawn to a novel xylose isomerase enzyme and recombinant yeasts which are able to utilize xylose.
2. Description of the Prior Art
Over 95% of U.S. fuel ethanol is produced using corn. Eventually, it is envisioned that annual corn ethanol production can expand to 12-15 billion gallons, consuming 31% or more of the corn harvest. For this reason, commercializing lignocellulose as a feedstock for further ethanol production has been made a national priority. Despite a growing commitment by industry to move towards these more challenging feedstocks, technical barriers still remain unsolved. One critical need is for more robust microbial strains capable of fermenting the more diverse mixture of neutral sugars released by hydrolysis of lignocellulose. Plant cell wall lignocellulose contains, in order of relative abundance, glucose, xylose, arabinose, galactose, and miscellaneous other sugars. While Saccharomyces strains ferment hexoses, they do not ferment the pentose sugars arabinose or xylose.
Several yeast, such as Pachysolen tannophilus, Scheffersomyces stipitis, and Candida shehatae naturally ferment xylose. While some of these are being pursued for commercialization, they have several defects, including the inability to grow anaerobically on xylose, low tolerance to acetic acid and other inhibitory chemicals common to biomass hydrolysates, and generally low productivity and yields compared to glucose-fermenting Saccharomyces cerevisiae. Attention in recent years has turned to engineering bacteria to selectively produce ethanol (Dien et al., 2003, Appl. Microbiol. Biotechnol., 63:258-266), improving the performance of native xylose-fermenting yeast (Jeffries, 2006, Curr. Opin. Biotechnol., 17:320-326), or engineering Saccharomyces strains to ferment pentose sugars, especially xylose (Hahn-Hägerdal et al., 2007 Appl. Microbiol. Biotechnol., 74:937-953; Van Maris et al., 2006, Antonie van Leeuwenhoek, 90:391-418; Karhumaa et al., 2005, Yeast, 22:359-368; Kuyper et al., 2004, FEMS Yeast Res. 4:655-664; and Kuyper et al., 2005, FEMS Yeast Res., 5:399-409).
Saccharomyces yeast can naturally utilize the pentose phosphate pathway intermediate xylulose. Genetic strategies to enable the yeast to ferment xylose have centered on introducing the needed activities for converting xylose to xylulose. Naturally xylose-fermenting yeasts convert xylose into xylitol using xylose reductase (XR) and xylitol into xylulose using xylitol dehydrogenase (XDH), but the process gives rise to a cofactor imbalance that results in production of xylitol (Van Maris et al., 2006, ibid; Kuyper et al., 2004, ibid). Saccharomyces yeast strains have been engineered that functionally express XR and XDH genes (Jeffries and Jin, 2004, Appl. Microbiol. Biotechnol., 63:495-509), and several have reasonable ethanol yields and reduced xylitol production, conceivably because enough oxygen enters the system to regenerate NAD+ from NADH via respiration instead of xylitol production (Karhumaa et al., 2005, ibid; Van Maris et al., 2006, ibid). Precisely controlled oxygen levels are nearly impossible to maintain in large-scale industrial operations, which limits the intermediate potential of these biocatalysts.
In an effort to convert xylose to xylulose without creating cofactor imbalances, Saccharomyces yeast strains were engineered to express a heterologous xylose isomerase (XI), which catalyzes this conversion directly (Karhumaa et al., 2005, ibid; Walfridsson et al., 1996, Appl. Environ. Microbiol., 62:4648-4651). However the activity of the XI enzyme was too low for efficient xylose metabolism. It was discovered that the xylose isomerase from Piromyces sp. E2 can be expressed at sufficient levels in S. cerevisiae (Harhangi et al., 2003, Arch. Microbiol., 180:134-141; Kuyper et al., 2004, ibid). After evolutionary engineering and expression of all genes encoding for the enzymes involved in the conversion of xylose into intermediates of glycolysis in addition to expression of XI and deletion of the gene encoding aldose reductase (to reduce production of the unwanted side product xylitol), a Saccharomyces strain was constructed that had an ethanol production rate of 0.46 g per g xylose per hour under anaerobic batch cultivation on xylose. When grown on 20 g per liter glucose and xylose each, an exponential glucose consumption phase followed by a slower, almost linear, xylose consumption phase was observed (Kuyper et al., 2005, ibid). Further selection for xylose growth yielded a strain that when cultivated in anaerobic batch culture with 20 g per liter glucose and xylose each, fermented all sugars in 24 hours, an improvement of 20 hours over the strain before selection. On xylose alone it had an ethanol production rate of 0.49 g per g xylose per hour under anaerobic batch cultivation (Van Maris et al., 2006, ibid). Growth in anaerobic xylose cultures is considered a highly desirable quality in industrial fermentation since it maintains cell viability and greatly increases the specific rate of ethanol production. Although uptake kinetics were also improved, the engineered Saccharomyces strains are only now moving towards commercialization (Hahn-Hägerdal et al., 2007, ibid). Co-fermentation of hexose and pentose sugars is still a major challenge.
More recently, Hughes et al. (U.S. patent application Ser. No. 12/568,071; 2008, Plasmid, 61:22-38) disclosed that recombinant S. cerevisiae produced by transformation of the yeast with heterologous polynucleotide sequences coding for XI and a xylulokinase (XKS) from Yersinia pestis exhibited high growth rates on media with xylose as the sole carbon source.
However, despite these and other advances, the need remains for improved enzymes and yeast for utilizing xylose.